I am investigating the mechanisms of mitochondrial polypeptide turnover in serum deprived maintenance cultures of a hepatoma cell line. By labeling cells with 3H- and 35S-methionine at the beginning and end of confluent maintenance, respectively, the rates of degradation of specific polypeptides in the membrane (t 1/2 = 48 h) and matrix (t 1/2 = 16 h) compartments can be investigated. The 3H/35S of polypeptides separated by two-dimensional electrophoresis or isolated by specific immunoprecipitation reflects their relative rate of degradation. Preliminary results reveal that the mitochondrial inner membrane is degraded heterogeneously whereas the subunits of major enzyme complexes are degraded at identical rates. I propose to accelerate the degradation of proteins in these cells by (1) addition of hormones, (2) depletion of a single amino acid from their complex media, and (3) substituting an arginine analogue for arginine. The first two conditions are thought to increase cellular protein degradation from basal to accelerated levels by stimulating the participation of lysosomes. The third condition produces abnormal polypeptides and will show if mitochondria have the capacity to rapidly recognize and remove proteins which have structural deviations from normal. These experiments will determine the extent to which mitochondria have the capacity to (1) be recognized by lysosomal degradation systems and, (2) degrade translation errors. The second part of the proposal focusses on compositional alterations in lysosomes during accelerated protein turnover. The alterations will be probed by crossed immunoelectrophoresis. Antiserum will be prepared against modified lysosome (tritosome) membrane and soluble antigens. The antiserum will be used in a second dimension to map major immunogens of the tritosome preparation by protein staining and enzyme characterization. These studies will then be extended to lysosome preparations from hepatoma cells to detect synthetic and post-translational differences in lysosome immunogens between cells undergoing basal or accelerated protein degradation. These studies should provide insight at the molecular level into the regulation of accelerated protein degradation and lysosome function.